蛋白免疫印迹法检测小分子蛋白的实验条件优化研究

目的:蛋白免疫印迹法自发明以来被广泛应用于现代生物学研究中的蛋白质定性和半定量分析。为了提高蛋白免疫印迹法的检测效率,需针对不同蛋白的特性调节相关的实验条件参数。本文旨在探讨免疫印迹法不同参数对小分子蛋白检测效果的影响,从而优化并获得最佳实验条件。方法:比较不同转膜电压和时间、转移缓冲液甲醇含量、不同化学发光剂对小分子蛋白的检测效果。结果:选择20 V、10 min转膜电压和时间所获得的信号显著高于10 V、25 min转膜条件,选择含20%甲醇转移缓冲液所获得的信号显著高于无甲醇转移缓冲液,选择飞克级化学发光剂所获得的信号显著高于纳克级化学发光剂。结论:选用高电压、短时间组合,选择含20%甲醇转移缓冲液和飞克级化学发光剂信号均有助于小分子蛋白免疫印迹检测。     

Superoxide dismutase in methylotrophic yeasts

Abstract Superoxide dismutase (SOD) activity of 4 strains of methylotrophic yeasts of the genera Candida, Torulopsis, Hansenula and Pichia was demonstrated. Strains tested were grown on glucose or methanol. Yeasts grown on methanol possessed considerably higher levels of SOD- and catalase activity than cells cultivated on glucose.     

Influence of pectinase treatment on fruit spirits from apple mash, juice and pomace

The current investigation was conducted to determine the influence of pectinase treatment on fruit spirits produced from apple mash, juice, and pomace. Crispin apples were processed into apple mash, juice, and pomace in our pilot-plant, and fermented with a commercial Red Star wine yeast (Sachharomyces cerevisiae Davis 904). After fermentation, the samples of fermented apple mash, juice, and pomace were distilled, and the distillates were analyzed by HPLC with a Bio-Rad Aminex HPX 87H column and a refractive index detector. Methanol, ethanol, n-propanol, iso-butanol, and iso-amyl alcohol were identified as the major alcohols in all the apple spirits. Student's t-test results indicate that there are significant differences between the methanol concentrations of pectinase treated and non-pectinase treated apple spirits. Duncan's multiple range tests show significant differences in the concentrations of methanol of the fruit spirits made from apple mash, juice, and pomace. Apple pomace yielded significantly higher methanol concentrations than apple mash and juice. Pectinase treatment had little effect on the concentrations of n-propanol, iso-butanol, and iso-amyl alcohol. It is concluded that fruit spirits made from the pectinase treated mash, juice, and pomace of Crispin apples had methanol concentrations significantly above the United States FDA guidance of 0.35% by volume or 280 mg/100 mL of fruit brandy containing 40% ethanol.     

Short-chained oxygenated VOC emissions in <Emphasis Type="Italic">Pinus halepensis</Emphasis> in response to changes in water availability

Short-chained oxygenated VOC (oxVOCs) emissions from Pinus halepensis saplings were monitored in response to changes in water availability. Online measurements were made with a proton transfer reaction—mass spectrometer under controlled conditions, together with CO₂ and H₂O exchange measurements. Masses corresponding to methanol and acetone were the most emitted oxVOCs. All the oxVOC exchanges, except that of acetone (M59), were significantly related to stomatal conductance and transpiration. Acetaldehyde (M45) emission showed, moreover, a strong dependence on the concentration of acetaldehyde in the ambient: stomatal opening (stomatal conductance above 75 mmol m⁻² s⁻¹) only allowed increased emissions when external concentration were below 6 ppb. Acetone (M59) presented an important peak of emission following light and stomatal opening in the morning when plants were water stressed. Thus, the alterations in oxVOC emissions in P. halepensis caused by the water deficit seem to be mainly driven by water stress effect on stomatal closure and oxVOC air concentrations.     

Roots of the wetland plants Typha latifolia and Phragmites australis are inhabited by methanotrophic bacteria in biofilms

Wetland plants create partly aerobic conditions in the rhizosphere by releasing oxygen to the waterlogged substrate. The present study was conducted to characterise the arrangement of rhizobacteria, especially those active in methane oxidation, in root-associated biofilms of wetland plants. Root cross sections sampled from Typha latifolia L. (broadleafed cattail) and Phragmites australis (Cav.) Trin. ex Steud. (common reed) were scanned using light and electron microscopy. Methane-oxidising bacteria were identified and quantified by immunological labelling of the α-subunit of the methanol dehydrogenase (α-MDH; encoded in mxaF). On roots of both species there was a diverse subset of bacteria arranged in a microbial biofilm around the roots’ exodermis. Similar bacterial densities in the root-associated biofilm were detected in more basal regions and closer to the root tip. Many microbes carried notable internal membrane systems that are characteristic of methanotrophic bacteria. This morpho-anatomical characterisation was confirmed by immunogold labelling with α-MDH antibodies. Quantification of labelled bacteria revealed that 34–43% of the biofilm bacteria were potentially capable of methane turnover. These findings confirm the presence of methane-oxidising bacteria in the root-associated biofilms of the two common macrophytes T. latifolia and P. australis. This implies that the methanotrophs participate essentially in the microbial processes related to oxygen-releasing roots of wetland plants.     

Optimization of protein solubilization for the analysis of the CD14 human monocyte membrane proteome using LC-MS/MS

Proteomic profiling of membrane proteins is of vital importance in the search for disease biomarkers and drug development. However, the slow pace in this field has resulted mainly from the difficulty to analyze membrane proteins by mass spectrometry (MS). The objective of this investigation was to explore and optimize solubilization of membrane proteins for shotgun membrane proteomics of the CD14 human monocytes by examining different systems that rely on: i) an organic solvent (methanol) ii) an acid-labile detergent 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), iii) a combination of both agents (methanol + PPS). Solubilization efficiency of different buffers was first compared using bacteriorhodopsin as a model membrane protein. Selected approaches were then applied on a membrane subproteome isolated from a highly enriched human monocyte population that was ~ 98% positive for CD14 expression as determined by FACS analysis. A methanol-based buffer yielded 194 proteins of which 93 (48%) were mapped as integral membrane proteins. The combination of methanol and acid-cleavable detergent gave similar results; 203 identified proteins of which 93 (46%) were mapped integral membrane proteins. However, employing PPS 216 proteins were identified of which 75 (35%) were mapped as integral membrane proteins. These results indicate that methanol alone or in combination with PPS yielded significantly higher membrane protein identification/enrichment than the PPS alone.     

Tryptophanase in aqueous methanol: the solvent effects and a probable mechanism of the hydrophobic control of substrate specificity

To shed light on the mechanism of hydrophobic control in reactions of microbial tryptophanase the direct effect of the solvent hydrophobicity on affinities of amino acid inhibitors was first examined. Values of inhibition constants (K_i) for a variety of amino acids were determined in 37.5% aqueous methanol, and no general correlation between the change of K_i, on passing from water to aqueous methanol, and amino acid hydrophobicity was found. The solvent effects on the separate stages of the external aldimine formation (K_D) and deprotonation to form a quinonoid intermediate (K_q) were determined for the reactions of tryptophanase with 2-oxindolyl- -alanine and -alanine by stopped-flow technique. For 2-oxindolyl- -alanine, which is a close transition-state analogue for the enzyme reaction with natural substrate, the decrease in the affinity in aqueous methanol is associated exclusively with the α-proton abstraction stage but not with the preceding formation of external aldimine. We conclude that the environment of amino acid side chains in the active site cannot be considered to be permanently hydrophobic irrespective of the bound amino acid. We suggest that complexes of tryptophanase with amino acids may exist either in a hydrophobic, presumably “closed”, conformation, where bound amino acids are isolated from the solvent, or in an accesible to solvent, “open”, conformation, depending on the structure of the bound amino acid and stage of the catalytic mechanism. For 2-oxindolyl- -alanine the transfer from an open to a closed conformation probably accompanies deprotonation of the external aldimine. The change of the active site hydrophobicity may provide an efficient way of modulating the relative acid–base properties of the catalytic groups to ensure the movement of protons in the “correct” direction depending on the elementary stage of catalysis.     

Cold-inducible selective degradation of peroxisomes in Hansenula polymorpha

Exposure of Hansenula polymorpha cells, grown in batch cultures on methanol at 37 degrees C, to a cold treatment (18 degrees C) is paralleled by a rapid degradation of peroxisomes present in these cells. Remarkably, the events accompanying organelle degradation at 18 degrees C are similar to those of selective glucose-induced peroxisome degradation in wild-type cells, described before. This observation was strengthened by the finding that cold-induced peroxisome degradation was not observed in mutants impaired in selective peroxisome degradation (Atg(-) mutants). Biochemical data indicated that the onset of peroxisome degradation was not triggered by the inactivation of peroxisome function due to the fall in temperature. We show that our findings have implications in case of fluorescence microscopy studies that are generally not conducted at physiological temperatures and thus may lead to strong morphological alterations unless proper precautions are taken.     

The use of flow cytometry in the evaluation of cell viability of cryopreserved sperm of the marine shrimp (Litopenaeus vannamei)

Although the cryopreservation of penaeid prawn sperm or embryos has definite applications in the aquaculture industry, there is no protocol routinely used for this procedure. One of the main problems relies on the limitations for the determination of sperm cell viability. In this study, we evaluated the toxicity and cryoprotectant effect of four agents, at three different concentrations, in sperm suspension, spermatic mass, and complete spermatophore of the marine shrimp Litopenaeus vannamei. Cells were frozen by fast and slow cooling rates. After thawing, they were analyzed by optical microscopy and flow cytometry, which was also utilized to determine spermatic viability by DNA staining with propidium iodine. Considering viability by morphotype analysis, the best result was obtained when the spermatic mass was frozen by slow cooling rate in the presence of methanol (61.6%). There was a positive correlation between morphotype analysis and flow cytometry, although the percentage of viable cells was always lower when determined by the later. These results show that flow cytometry is a valuable tool to evaluate sperm cell viability in decapod species and it is more sensitive technique than optical microscopy.     

The energy metabolism of <Emphasis Type="Italic">Methanomicrococcus blatticola</Emphasis>: physiological and biochemical aspects

Methanomicrococcus blatticola, a methanogenic archaeon isolated from the cockroach Periplaneta americana, is specialised in methane formation by the hydrogen-dependent reduction of methanol, monomethyl-, dimethyl- or trimethylamine. Experiments with resting cells demonstrated that the capability to utilise the methylated one-carbon compounds was growth substrate dependent. Methanol-grown cells were unable of methylamine conversion, while cells cultured on one of the methylated amines did not metabolise methanol. Unlike trimethylamine, monomethyl- and dimethylamine metabolism appeared to be co-regulated. The central reaction in the energy metabolism of all methanogens studied so far, the reduction of CoM-S-S-CoB, was catalysed with high specific activity by a cell-free system. Activity was associated with the membrane fraction. Phenazine was an efficient artificial substrate in partial reactions, suggesting that the recently discovered methanophenazine might act in the organism as the physiological intermediary electron carrier. Our experiments also showed that M. blatticola apparently lacks the pathway for methyl-coenzyme oxidation to CO₂, explaining the strict requirement for hydrogen in methanogenesis and the obligately heterotrophic character of the organism.